Diabetes Mellitus (DM) of type I (DM I) and type II (DM II) causes significant cardiovascular disease including diabetic cardiomyopathy (DC) and resulting in heart failure (HF) which presents a growing healthcare problem. Mechanisms which cause DC and DM related HF are only incompletely explored. In Aim I we pursue novel findings showing that mitochondrial (Mito) proteins in diabetic (D) hearts exhibit excessive enzymatic O-linked glycosylation (O-GlcNAc), which is linked to diminished Mito complex function and a diminished ability to perform maximal cardiac work. Exposing cardiac myocytes (CM) to high glucose (HG) results in similar Mito changes. Viral vector based expression of the O-GlcNAcase enzyme (GCA) in CM of D hearts markedly improves Mito complex function and maximal cardiac function in spite of persistent DM. We also generated transgenic (Tg) mice with tetracycline (Tet) transactivator mediated conditional expression of (GCA) in CM. GCA expression in CM of D hearts results in decreased protein O-GlcNAc of Mito DNA encoded subunit I of complex IV. In Aim II we pursue our novel findings that in CM exposed to high glucose (HG) markedly increased Mito fission and decreased Mito fusion occurs contributing to diminished Mito function. In CM of DM hearts increased Mito fission is also identified. In addition in DM hearts and in CM exposed to HG the fusion related protein OPA1 is decreased and excessively O- GlcNAcylated, whereas the Mito fission related proteins Fis1 and Drip1 are markedly increased. Preliminary results indicate that normalizing OPA1 levels and its O-GlcNAc status, using viral vector based expression of OPA1 or GCA transgenes (tge) in CM exposed to HG returns Mito fusion towards normal. Studies of Aim III are directed at increasing the activity of the pyruvate dehydrogenase complex (PDC) of the D heart and enhancing glucose oxidation (ox). Our results show, that inhibiting in the activity of PD kinase (PDK) 2 in the D heart using a viral vector expressed dominant negative PDK2 mutant, returns the decreased glucose ox to normal, markedly diminishes the excessive FA ox and improves the energetic efficiency of cardiac contraction. In an alternative approach to return D heart fuel flux to normal we will pursue our preliminary results that a shRNA directed at FA transporter 6 markedly diminishes its level in CM. These studies are conducted in DM I and DM II models. Novel insights and approaches to improve the function of the D heart in spite of the persistence of the D milieu may result. PUBLIC HEALTH RELEVANCE: Diabetes mellitus is an increasingly important public health problem and leads to decreased function of the heart termed diabetic cardiomyopathy. Exposure to high glucose contributes to decreased cardiac function and the mechanisms which lead to these alterations are explored in this application. The identification of new targets to improve the function of the diabetic heart may result from this research.